尿石素A抑制BMMs向破骨细胞分化

Urolithin A Inhibits the Differentiation of BMMs into Osteoclast

目的探究尿石素A对小鼠骨髓来源巨噬细胞(bone marrow derived macrophages,BMMs)向破骨细胞分化的影响。方法提取小鼠骨髓腔BMMs,通过CCK-8法检测尿石素A对BMMs的增殖毒性,使用核因子-κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)诱导BMMs向破骨细胞分化并与浓度梯度尿石素A共培养,抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色检测破骨细胞数量和面积大小,实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)法检测破骨分化相关基因水平表达,2′,7′-二氯二氢荧光素二乙酸酯(2′,7′-Dichlorofluorescin diacetate,DCFH-DA)检测细胞活性氧(reactive oxygen species,ROS)水平。结果CCK-8法检测结果显示,尿石素A在16μmol/L范围内对BMMs无明显毒性;TRAP染色结果表明,尿石素A显著抑制了BMMs向破骨细胞分化,成熟破骨细胞数目和面积减少(P<0.01);RT-qPCR检测结果显示,尿石素A明显抑制了破骨分化相关基因(C-FOS、CTSK、MMP9、ACP5)的相对表达水平(P<0.01);DCFH-DA检测结果显示,尿石素A明显降低了RANKL诱导的高ROS水平。结论尿石素A能抑制RANKL诱导的BMMs向破骨细胞分化,并降低分化过程中的ROS,有望作为骨质疏松治疗的潜在药物。

Objective To investigate the effect of urolithin A on differentiation of mice bone marrow derived macrophages(BMMs)into osteoclast.Methods The BMMs were extracted from the bone marrow cavity of mice.The proliferation toxicity of urolithin A to BMMs was detected by CCK-8 method.Receptor activator of nuclear factor-κB ligand(RANKL)induced osteoclast differentiation of BMMs and co-cultured with concentration gradient urolithin A.Tartrate resistant acid phosphatase(TRAP)staining to detect the number and area size of mature osteoclasts.The expression of osteoclast differentiation-related genes was detected by real-time quantitative polymerase chain reaction(RT-qPCR).2′,7′-Dichlorofluorescin diacetate(DCFH-DA)was used to detect the cellular ROS levels.Results CCK-8 test results showed that no obvious toxicity of urolithin A to BMMs in the range of 16μmol/L.The TRAP staining results showed that urolithin A significantly inhibited the differentiation of BMMs into osteoclast,and the number and area of mature osteoclasts decreased(P<0.01).RT-qPCR test results showed that urolithin A significantly inhibited the relative expression of osteoclast differentiation-related genes(C-FOS,CTSK,MMP9,ACP5)(P<0.01).DCFH-DA fluorescence assay test showed that urolithin A significantly decreased RANKL-induced high ROS.Conclusion Urolithin A can inhibit RANKL-induced differentiation of BMMs into osteoclast and decrease the ROS during differentiation,which may be a potential drug for osteoporosis treatment.